Toxic shock syndrome toxin-1 (TSST-1), the potent staphylococcal exopr
otein linked to most cases of the toxic shock syndrome, is a V-beta-re
stricted T-cell mitogen (a so-called ''superantigen''). TSST-ovine (TS
ST-O) is a natural variant of TSST-1, and is produced by certain ovine
mastitis-associated strains of Staphylococcus aureus. Compared to TSS
T-1, TSST-O is only weakly mitogenic for leporine or murine splenocyte
s. It differs from TSST-1 at 7 amino acid residues over its 194 amino
acid length. Terminus shuffling between the two proteins has suggested
that their C-terminal differences (T69, Y80, E132, and I140 in TSST-1
; I69, W80, K132, and T140 in TSST-O) are in part responsible for thei
r discrepant mitogenic properties. In order to explore further the fun
ctional consequences of altering TSST-1 at residues 132 and 140, we en
gineered point mutants of TSST-1 at those positions. The mutant protei
ns were purified to homogeneity from culture supernatants of a nontoxi
genic strain of S. aureus using a combination of ultrafiltration, liqu
id-phase isoelectric focusing, and ion-exchange chromatography. The mu
tants retained global structural integrity as evidenced by circular di
chroism spectroscopy, their preserved resistance to trypsin digestion,
and their preserved binding to a neutralizing murine monoclonal antib
ody. The mutants were then tested for mitogenicity for human T-cells:
The mutant I140T was approximately as active as wild-type TSST-1, whil
e the mutant E132D was about 10-fold attenuated. On the other hand, th
e mutants E132A or E132K were each at least 1000-fold attenuated. We c
onclude that the mitogenic activity of TSST-1 for human T-cells depend
s critically on the presence and precise position of the negative char
ge at residue 132.